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Title: Study of SNAI1 gene impact on differentiation of induced pluripotent stem cells into skeletal muscle in the context of rhabdomyosarcoma development
Abstract:
Gene expression modulation with genome editing (GE) tools and RNA interference technology is widely used in tumor research. GE utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALEN) tools to generate precise double-strand breaks in the DNA structure. Induced repair mechanisms may lead to gene knockout. shRNA leads to the gene knockdown as the result of the interaction with the targeted transcript. In the current study, three different methods for SNAI1 knockout or knockdown in rhabdomyosarcoma (RMS) cells were compared The aims of the study were to design CRISPR/Cas9 and TALEN systems targeting SNAI1 and to compare their effectiveness and biological effects with shRNA. Therefore, CRISPR/Cas9, TALEN and shRNA tools were introduced separately to the cells. Next, the genome sequence, transcript levels, and protein expression levels of SNAI1 were evaluated. The biological effects of SNAI1 expression modulation were accompanied by changes in the morphology of the cells and modulation of the expression of myogenic factors (MYOD, MYOG, MHC, MSTN and MEF2A) and HDAC1. We confirmed the similar effectiveness of the tested methods. Nevertheless, the low efficiency of the GE tools was a limiting factor in obtaining biallelic gene knockouts. To conclude, we established and characterized thr ; ee different methods of SNAI1 expression modulation: knockout with GE tools and knockdown with shRNA technology. These results will be used in further studies investigating the role of SNAI1 in RMS. Induced pluripotent stem (iPS) cells with their ability to differentiate into three germ layers constitute a perfect tool to study human embryo development processes such as myogenesis. Currently, many protocols describing the differentiation of iPS cells into myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. The aim of the current study was to verify and compare, side by side, three different protocols of skeletal muscle differentiation with the characteristics of the obtained cells. Therefore, iPS cells were differentiated using protocols previously described in the literature. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II started with strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF. Protocol III utilizes monolayer cell culture in three special media, leading to WNT activation and TGF-β and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic ; regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol in different time points. Obtained cells were characterized by the presence of the extracellular markers CD10, CD24 and CD56 using flow cytometry. Cell fusion ability and vimentin presence with immunofluorescence were verified. Our results revealed that protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation. Obtained results will be used in further studies investigating the role of SNAI1 in RMS.
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Rada Dyscypliny Nauki medyczne
Promoter:
Majka, Marcin ; Skrzypek, Klaudia
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Last modified:
Nov 22, 2023
In our library since:
Nov 22, 2023
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All available object's versions:
http://dl.cm-uj.krakow.pl:8080/publication/4995
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| Edition name | Date |
|---|---|
| ZB-137549 | Nov 22, 2023 |
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